This invention relates to amplification assays to detect nucleic-acid targets.
This application relates to assays employing exponential amplification of a target sequence. The target sequence may be RNA or DNA. By xe2x80x9camplification of a target sequence,xe2x80x9d we mean to include amplification of the target sequence itself and also amplification of a transcript thereof, as when an RNA target sequence is amplified by first creating a DNA transcript with reverse transcriptase and then amplifying the DNA transcript. By xe2x80x9cexponential amplificationxe2x80x9d we mean an amplification reaction or reactions that generate products (xe2x80x9campliconsxe2x80x9d) that include both plus strands and complementary minus strands.
In referencing target sequences, control sequences, amplicons and probes, we mean to include both plus and minus strands. Thus, it will be understood that when referring to cross hybridization of a control sequence with a target sequence or to cross hybridization of two target sequences during amplification, we are referring to hybridization of the plus strand of one to the minus strand of the other. Similarly, when we refer to hybridization of a probe to a target sequence, we mean to include hybridization of the probe to either the plus-strand or the minus strand of the target sequence itself or to an amplicon, that is a plus-strand copy or minus-strand copy of the target sequence.
Several reaction schemes are used in assays employing amplification of a target sequence. The most widely used is the polymerase chain reaction (PCR) process. It will be used herein for presentation of the specifics of the prior art and the specifics of this invention. However, it will be understood that this invention also applies to other reaction schemes, including nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR) (Guatelli et al. (1990)) and strand displacement amplification (SDA) (Walker et al. (1992)).
The polymerase chain reaction process is well known. It is the most widely used technique for amplifying DNA and RNA (RT-PCR) targets, including amplification as part of assays to detect the presence of DNA and RNA targets for many purposes, including, for example, in vitro diagnostics, genetic analyses, forensics, food and agricultural testing, and parentage testing. PCR is used for detection even at the level of a single cell (in situ PCR).
Quantitative PCR assays are also well known. Quantitative PCR assays for DNA and RNA have been widely used to study disease processes (see, for example, Clementi et al., 1993). One type of quantitative PCR assay involves simultaneously amplifying control molecules and samples containing (or suspected to contain) a target sequence. Receptacles containing known amounts of control molecule are thermally cycled with receptacles, most commonly tubes or wells or slides, containing the unknown amount of target. In addition to the pair of PCR primers for the target, a pair of PCR primers is required for each control molecule. Following amplification, the amounts of amplified products (amplicons) are compared. See generally Clementi el al., 1994 and Kahn et al., 1992. Partly due to variation in amplification efficiency among primers, only relative quantitation between samples is possible.
Another type of quantitative PCR assay is quantitative-competitive PCR (QC PCR). In this method a control molecule which is similar to but ultimately distinguishable from the target sequence competes with the target sequence for the same pair of primers. Following competitive amplification, the two products synthesized (amplicons) are distinguished, for example, by size using gel electrophoresis. See generally Wang et al., 1989 and Becker-Andre, 1991. While permitting more than relative quantitation between samples, QC PCR has inherent disadvantages and limitations. Post-amplification manipulation is required. This complicates the assay, decreases throughput, increases labor, and risks contamination of untested samples by amplicon carryover. Assay design is complicated by the need for a competitor-control that amplifies with an efficiency very close to that of the target unknown. For reasonable quantitation, most QC PCR assays are performed in multiple tubes containing serial dilutions, of the competitor-control, typically five-fold dilutions, but in some assays two-fold dilutions for better accuracy. Differences in amplification efficiency between the target and the competitor-control usually compel analysis during the exponential phase of amplification, because errors become too large during the subsequent linear phase. (Mullis and Faloona, 1987). Precision is limited, varying a minimum of fifty percent between parallel assays.
A more recently developed type of quantitative PCR assay has been called the 5xe2x80x2-nuclease assay and xe2x80x9creal-time PCR.xe2x80x9d See generally, Gibson et al., 1996; Heid et al., 1996; Gelfand et al., 1993; and Livak et al., 1996. This method utilizes detector probes that are linear DNA sequences labeled with two different fluorescent dyes, for example, a reporter dye such as FAM and a quenching dye such as TAMRA. Commercial kits from the Applied Biosystems Division of The Perkin-Elmer Corporation (Foster City, Calif. (U.S.A.)) are available under the trademark TaqMan(trademark). When not hybridized to target (original unknown or amplicon) the quenching dye partially quenches the reporter dye. During the annealing step of a PCR cycle, the probes hybridize to the target sequence, and during the extension step of the PCR cycle, the probes are cleaved by the 5xe2x80x2xe2x86x923xe2x80x2 nucleolytic activity of DNA polymerase. Cleavage releases the reporter dye from the quenching dye, resulting in an increase in fluorescence. Fluorescence can be monitored throughout the PCR amplification. An instrument available from the Applied Biosystems Division of The Perkin-Elmer Corporation, the ABI PRISM 7700, monitors fluorescence in 96 tubes simultaneously in real time. An improved probe suitable for real-time PCR has been developed. See Tyagi and Kramer, 1996. This probe, referred to as a xe2x80x9cmolecular beaconxe2x80x9d, possesses a stem-and-loop structure, has a higher signal-to-background ratio than linear probes and also has improved allele discrimination. During the annealing step of a PCR cycle, molecular beacon probes hybridize to the target sequence and fluoresce, but during the extension step of the PCR cycle, the probes leave the target and do not interfere with polymerization.
Due to sample-to-sample variations in PCR efficiency, only data from the early, exponential amplification phase should be used. That limited data permits a determination of the PCR cycle number at which fluorescence becomes detectable above background (the cycle threshold). The cycle threshold decreases in proportion to the logarithm of initial target concentration. A standard curve can be generated from the cycle thresholds of a dilution series of known starting concentrations of target, and the cycle threshold of a sample containing an unknown amount of target sequence can be compared to the standard curve in order to determine the amount of target sequence present in the sample. Real-time PCR has a wider dynamic range than QC PCR. Importantly, it does not suffer from the serious disadvantages resulting from opening tubes after amplification. It utilizes homogeneous detection with a probe that is added prior to amplification. Nevertheless, accuracy is limited due to variations in amplification efficiency. For example, Gibson et al. (1996) performed real-time PCR using two sets of tubes. Each set contained triplicate two-fold dilutions of a control molecule and a fixed amount of unknown. Despite use of averaged triplicate samples of two-fold dilutions to create a standard curve for cycle thresholds and use of averaged triplicate samples of unknown, quantitation of the unknown in the two separate experiments differed by thirty percent.
An aspect of this invention is nucleic acid hybridization assays that do not require post-amplification manipulation, that include at least two sequences which are subject to the same reaction kinetics, and that include homogenous detection utilizing interactively labeled hybridization probes.
Another aspect of this invention is quantitative, homogeneous PCR assays wherein the precision is significantly improved over the thirty-percent variability of real-time PCR.
Another aspect of this invention is homogenous nucleic acid hybridization assays, including especially PCR assays, to detect amounts of two co-amplifiable, cross hybridizable targets in a sample, either the relative amount of one to the other or absolute amounts of both, with high precision.
These and other aspects of this invention will be apparent from the description, including the figures, which follow.
Two or more different sequences that cross hybridize, as during the annealing step of a PCR reaction, can be co-amplified using a single set of primers. By xe2x80x9ccross hybridizexe2x80x9d we mean that the amplicons of each sequence hybridize not only to themselves but also to amplicons of the other sequences. For such sequences, the amplifications of the sequences are linked; they follow the same reaction kinetics and act as a single amplicon. We refer to this as non-competitive amplification. It differs from competitive amplification, such as in QC PCR (wherein two sequences compete for a single set of primers and follow different reaction kinetics, and where one sequence may grow at the expense of the other). The difference is profound with respect to quantitation and relative quantitation in homogeneous detection assays in which tubes are not opened for further manipulation following amplification.
Detection in the assays of this invention utilizes what we refer to as xe2x80x9cdual-labeled hybridization probesxe2x80x9d by which we mean hybridization probes having at least two interactive labels, whose signal varies depending on whether the probe is hybridized to a strand or free-floating in a single-stranded conformation. Fluorescent labels are preferred and will be used to illustrate and explain such probes. Such probes are suitable for homogeneous assays, because separation of bound probes from unbound probes is not required, as is required when traditional fluorescently labeled probes not having interactive labels are used. As stated above, we are aware of two different types of dual-labeled hybridization probes. One is a linear probe known as the TaqMan(trademark) probe, described in Heid et al., 1996; Gibson et al., 1996; Livak et al., 1996; and Gelfand et al., 1993; all of which are incorporated herein in their entireties. The probe is a linear oligonucleotide complementary to a non-primer portion of a sequence to be amplified (that is, it hybridizes between the primers, for example, PCR primers). The probe is labeled at two nucleotides removed from each other with a reporter dye such as FAM and a quenching dye such as TAMRA. When the probe is not hybridized to a strand, the quencher partially quenches the fluorescence of the reporter. When the probe hybridizes to the target sequence, as during the annealing step of PCR, it sits in the path of the DNA polymerase that will generate a copy, as in the extension step of PCR. The DNA polymerase cleaves the probe, thereby permanently severing the reporter from the quencher. Such probes have a limited ability to xe2x80x9callele discriminate,xe2x80x9d by which we mean to distinguish between two sequences that differ by as little as a single nucleotide.
A second type of dual-labeled hybridization probe useful in assays according to this invention is a hairpin probe in which a probe sequence is a loop and flanking arm sequences form a double-stranded stem. Each arm contains one of the at least two interactive labels, typically a fluorophore and a quencher. Fluorescently labeled molecular beacons undergo a fluorogenic conformational change when they hybridize to their target. A fluorescent moiety is covalently linked to the end of one arm and a quenching moiety is covalently linked to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher is a non-fluorescent chromophore that emits the energy that it receives from the fluorophore as heat, fluorescence does not occur. When the probe encounters a target molecule, it forms a probe-target hybrid that is longer and more stable than the stem hybrid. The rigidity and length of the probe-target hybrid precludes the simultaneous existence of the stem hybrid. Consequently, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem hybrid to dissociate and the fluorophore and the quencher to move away from each other, restoring fluorescence. Fluorescence increases as much as 900-fold when these probes bind to their target. Various label pairs may be used, including among others the fluorophore EDANS and the quencher DABCYL. These probes, called xe2x80x9cmolecular beaconsxe2x80x9d, and their preparation and use in homogenous, real-time PCR assays are described in Tyagi and Kramer, 1996, and in Tyagi et al., 1996, each of which is incorporated herein in its entirety. The ability of a molecular beacon probe containing a probe section 15 nucleotides long flanked by complementary arms, each 5 nucleotides long, to effectively discriminate between targets differing by a single nucleotide is described. We prefer molecular beacon probes for use as dual-labeled probes in assays according to this invention. Specifically we prefer fluorescently labeled molecular beacons having a probe sequence 7-25 nucleotides in length and flanking arm sequences 3-8 nucleotides in length.
One embodiment of an assay utilizing non-competitive amplification of a target sequence, preferably PCR amplification, is a quantitative assay for an unknown amount of a target sequence. A co-amplifier DNA strand which will cross hybridize with the target sequence is utilized in known amount. We sometimes refer to this as a control molecule. A probe specific for the target sequence and a probe specific for the co-amplifier are used. Preferred probes are fluorescer/quencher-labeled molecular beacon probes (Tyagi and Kramer (1996)) which are capable of discriminating against a single base-pair mismatch. With these probes, the control molecule may be identical to the target sequence except at one nucleotide. Other dual-labeled probes whose signal is a function of the amount of target (original target sequence or amplicon), such as TaqMan(trademark) probes described above, can also be used. In many instances a series of PCR amplification reactions containing a dilution series of the control molecule DNA will be used.
We have discovered that the ratio of the target sequence to the control molecule is constant throughout the PCR reaction, including in the linear phase. Thus, the ratio can be determined at any cycle in which the fluorescences of both probes are above the background level. More preferably, the ratio can be determined at many cycles and averaged to provide an extremely accurate quantitation. Persons in the art will understand how to calculate the ratios.
Various combinations of probes can be employed, for example: a probe specific for the control (preferably complementary to a portion of the control sequence that differs by at least one nucleotide, preferably one nucleotide, from the corresponding portion of the target sequence of the unknown) and a probe specific for both the unknown and the control; a probe specific for the unknown and a probe specific for both the unknown and the control; a probe specific for the control and a probe specific for the unknown; and a probe specific for the control, a probe specific for the unknown and a third probe specific for both the control and the unknown (obviously complementary to a sequence that occurs in both the control and the unknown). In each of these embodiments one obtains a ratio or ratios from which the concentration of unknown target can be readily calculated. Especially preferred is the use of three probes: one specific for the unknown target sequence, one specific for the control, and one specific for both, that is, designed to hybridize equally to both the target and the control. Use of three probes provides additional data and an internal control. An instrument such as the ABI PRISM 7700, described above, can be programmed to make computations automatically. However, the method is so accurate that a single reading during, or at the end of, the PCR amplification can be used.
Another embodiment of non-competitive PCR assays is an assay to detect the ratio of two closely related target sequences, such as alleles or mutants, for example, drug-resistant mutant pathogens. Amplicons containing genetic alleles differing by a single nucleotide and amplicons containing drug-resistant mutants differing from a wild-type pathogen, for example bacterium or virus, by a single nucleotide will cross hybridize as required for non-competitive amplification. When two unknowns co-amplify in a non-competitive PCR reaction, the ratio of the progeny amplicons derived from the first unknown to progeny amplicons derived from the second unknown remains constant throughout the amplification, including the linear phase. We have shown, for example, that one can distinguish as little as two percent of mutant DNA in an otherwise wild-type DNA population. It will be appreciated from the preceding discussion that this embodiment can be precisely quantitative by using a control in a known amount.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.
Other features and advantages of the invention, e.g., accurate quantitation of pathogens in patients will be apparent from the following detailed description, from the drawings and from the claims.